1
Q
Mutations
A
- alterations in the DNA structure that can produce permanent change in the genetic information encoded if they are not repaired
- ex. Glu6 -> Val in hemoglobin -> sickle cell anemia
- most cells have only 1 or 2 sets of genomic DNA, so damage can be disastrous unless its repaired
- mutations can be silent if they are in non-essential regions of the DNA, or have negligible effect on the gene
- rarely mutations will confer a biological advantage - > adaptation and evolution
2
Q
Two types of mutations
A
- Point mutations - substitution of one base pair for another (usually caused by modification/damage of bases)
- Insertions/deletions - of one or more bases (indels) - often generated by “DNA intercalating” agents
3
Q
Point mutations cause base substitutions
A
- base mismatches often occur when the template base has been modified (damaged) so that its H-bond donor/acceptor arrangement now favors a base other than the one it normally pairs with
- if this damage isn’t corrected, the damaged template strand will direct an incorrect base to incorporate into the daughter strand during DNA replication
4
Q
Types of base substitution
A
- the most common base modifications result in replacement of one purine by another purine or of one pyrimidine by another pyrimidine - transition
- less frequently a purine is replaced by a pyrimidine or vice versa - transversion
5
Q
Deamination
A
- removes an amino group
- deaminating agents - nitrous agents (deaminates aromatic primary amines)
- treatment of DNA with nitrous acid results in deamination of adenine, conversion to hypoxanthine
- deamination induces a mutation from A-T to G-C
- deamination can also occur spontaneously
- deaminated base pairs are repaired by base excision repair
6
Q
Chemical mutations that cause mutations
A
- deamination
- alkylating agents
- intercalation agents
- UV and other ionizing radiation damage
- oxidative damage to DNA by reactive oxygen species
7
Q
Alkylating agents
A
- add a methyl or ethyl group usually to purines
- usually the modification/damage is repaired before a mutation occurs - direct repair by the DNA repair enzyme O6-alkylguanine alkyl transferase, which transfers the methyl group to a cysteine in its active site
- the enzyme becomes permanently alkylated
8
Q
Intercalation agents
A
- flat aromatic molecules such as acridines can intercalate between adjacent base pairs in the DNA double helix
- their presence can lead to the insertion or deletion of one or more base pairs
- EthBr is a intercalating agent used in the lab to visualize DNA on agarose gel
9
Q
UV and other ionizing radiation damage to DNA
A
UV light can be absorbed by these bases and you have adduct
- Reaction cannot be reversed
- DNA is damaged
- thymine dimers are made
More exposure = less likely to deal with damage
10
Q
Explain repair of UV-induced thymine dimers in bacteria and plants
A
- thymine dimers block replication and transcription because the helix distortion (kink) blocks the polymerization machinery
- in bacteria, this “lesion” can be corrected by a photo reactivating enzyme photolyase that binds specifically to thymine dimers and uses visible light to hydrolyse the bonds linking the pyrimidine rings
- the ability of an organism to survive UV irradiation directly correlates with its ability to remove thymine dimers from its DNA
- photo-reactivation is a method of direct repair of DNA, occurs in bacteria and lower eukaryotes (including plants)
- human cells do not seem to contain photolyases, thymine dimers are repaired by nucleotide excision and in some cases base excision repair
- unrepaired thymine dimers can lead to melanoma (skin cancer) in humans
11
Q
General Mechanisms of DNA repair
A
- direct repair - damaged bases are not removed but repaired on site
- base excision repair - offending base is removed and replaced
- nucleotide excision repair - offending nucleotides are removed and replaced
- mismatch repair - when the wrong nucleotide is added during DNA replication, system must discriminate between the template (correct) and the newly synthesized strand (incorrect); many enzymes involved
- homologous recombination - repair of double strand breaks, stalled replication
12
Q
Base excision repair
A
- when a base of a nucleotide is damaged (cytosine)
1. Cytosine can easily lose an amino group, forming a base called uracil
2. uracil cannot form a base pair with guanine
3. an enzyme, glycosylase, discovers the defect and excises the base of uracil. the glycosylase hydrolyzes the glycosidic bond between the base and the sugar
4. another couple of enzymes remove the rest of the nucleotide from the DNA strand
5. DNA polymerase fills in the gap and the DAN strand is sealed by DNA ligase/ATP hydrolysis
13
Q
Nucleotide excision repair
A
- UV radiation can make 2 thymines bind to each other incorrectly
- the enzyme exinuclease finds the damage and cuts the DNA strand. 12 nucleotides are removed
- DNA polymerase fills in the resulting gap
- DNA ligase seals the DNA strand
